Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Le vélo électrique: un outil pour la santé ou un gadget "écolo" [Electrically assisted bicycles: health enhancement or "green" gadget?].

Electrically assisted bicycles (EAB) are flourishing in cities throughout the world and capitalize on ecological and practical advantages, helping in the fight against pollution, CO2 emissions and traffic jam. Human power is necessary to activate the electrical support, so that it equals to a moderate intensity physical activity (> 3 MET), or a vigorous one on hilly courses (>6 MET). The ecological benefits are obvious and transportation departments tend to support citizens who purchase one. EAB offer increased mobility at speeds of 15 to 25 km/h depending on hills and fitness of the rider, but could cause more accidents. EAB is linked to a real physical activity beneficial for health, but potentially more dangerous than a traditional bicycle.

Polypharmacy as predictor of drug cost, length of hospital stay and mortality rate in nursing homes : a retrospective analysis of pharmacy medication records

Background and objectives: Polypharmacy (PP) is a typical con-sequence of multiple chronic conditions in elderly patients. PP is commonly defined as the use of multiple concurrent drug therapies although a standard definition is not used. The aims of this study were to assess the PP rate among nursing home (NH) residents using the data of the pharmacy medication records and to investigate the threshold level of PP as predictor of drug cost, length of hospital stay and mortality rate

Ultrastructural changes of the internal limiting membrane removed during indocyanine green assisted peeling versus conventional surgery for idiopathic macular epiretinal membrane

Résumé : Introduction : L'objectif de cette étude était d'une part d'évaluer les caractéristiques histologiques des fragments cellulaires rétiniens attachés à la limitante interne après vitrectomie et pelage d'une membrane epirétinienne, et d'autre part de mettre en évidence des différences histologiques entre les cas opérés avec ou sans l'aide d'ICG dilué dans du glucose 5%. Méthodes Nous avons examiné rétrospectivement l'histologie de 88 spécimens de membranes épimaculaires contenant la limitante interne de la rétine, qui ont été enlevés chirurgicalement entre 1995 et 2003. L'analyse histologique a centré principalement l'attention sur la présence et les caractéristiques des fragments cellulaires rétiniens attachés à la limitante interne. L'analyse statistique a comparé les résultats entre le groupe I (chirurgie conventionnelle sans l'aide de l'ICG) et le groupe II (chirurgie à l'aide de l'ICG). Résultats Soixante et onze patients ont eu une vitrectomie sans l'aide de l'ICG (groupe I) et 17 avec l'aide de l'ICG (groupe II). Le nombre de débris de cellules de Müller à la surface rétinienne de la limitante interne était plus important dans le groupe I (sans ICG) que dans le groupe II (avec ICG) (40.8% versus 11.8% ; p = 0.024). Des larges fragments cellulaires rétiniens attachés à la limitante interne ont été plus fréquemment observés dans le groupe I (sans ICG) que dans le groupe II (avec ICG) (63.4% versus 23.5%; p= 0.003). Dans cinq (7%) cas du groupe I, de gros éléments cellulaires rétiniens ont été mis en évidence (des axones neuraux ou des vaisseaux sanguins). De tels éléments n'ont pas été retrouvés dans les spécimens du groupe II (avec ICG). Conclusions L'utilisation de l'ICG dilué dans du glucose 5% pour faciliter le pelage d'une membrane épimaculaire et notamment l'ablation de la limitante interne de la rétine semble diminuer de manière significative le nombre et la taille des débris des cellules de Muller adhérents à la face rétinienne de la membrane limitante interne de la rétine. Cette observation suggère que l'utilisation per-opératoire d'ICG dilué dans du glucose 5% facilite l'ablation de la limitante interne pendant la chirurgie de la membrane epirétinienne en diminuant l'adhérence de la limitante interne à la rétine.

Ultrastructural changes of the internal limiting membrane removed during indocyanine green assisted peeling versus conventional surgery for idiopathic macular epiretinal membrane.

PURPOSE: To evaluate the histologic features of cellular retinal fragments on the internal limiting membrane (ILM) removed during idiopathic macular epiretinal membrane (MEM) peeling surgery with and without the aid of indocyanine green (ICG) diluted in 5% glucose. METHODS: ILM specimens removed from 88 eyes during idiopathic MEM surgery between 1995 and 2003 were reviewed retrospectively. Histologic analysis focused on the presence and characteristics of retinal fragments on the retinal surface of the ILM. Statistical analysis compared the results between group I (conventional surgery) and group II (ICG-assisted peeling). RESULTS: Seventy-one eyes underwent MEM surgery without the aid of ICG (group I) and seventeen underwent MEM ICG-assisted surgery (group II). The amount of Müller cell debris on the retinal surface of the ILM was more significant in the group I than in the group II (40.8 vs. 11.8; P = 0.024). Large fragments of Müller cells were more frequently observed in the group I (no ICG) than in the group II (ICG) (63.4 vs. 23.5%; P = 0.003). CONCLUSIONS: The use of ICG diluted with 5% glucose in ILM removal during MEM surgery was associated with less retinal debris attached to the retinal face of the ILM compared with surgery in which ICG was not used.

A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads.

In sharp contrast to birds and mammals, most cold-blooded vertebrates have homomorphic (morphologically undifferentiated) sex chromosomes. This might result either from recurrent X-Y recombination (occurring e.g. during occasional events of sex reversal) or from frequent turnovers (during which sex-determining genes are overthrown by new autosomal mutations). Evidence for turnovers is indeed mounting in fish, but very few have so far been documented in amphibians, possibly because of practical difficulties in identifying sex chromosomes. Female heterogamety (ZW) has long been established in Bufo bufo, based on sex reversal and crossing experiments. Here, we investigate a sex-linked marker identified from a laboratory cross between Palearctic green toads (Bufo viridis subgroup). The F(1) offspring produced by a female Bufo balearicus and a male Bufo siculus were phenotypically sexed, displaying an even sex ratio. A sex-specific marker detected in highly reproducible AFLP genotypes was cloned. Sequencing revealed a noncoding, microsatellite-containing fragment. Reamplification and genotyping of families of this and a reciprocal cross showed B. siculus to be male heterogametic (XY) and suggested the same system for B. balearicus. Our results thus reveal a cryptic heterogametic transition within bufonid frogs and help explain patterns of hybrid fitness within the B. viridis subgroup. Turnovers of genetic sex-determination systems may be more frequent in amphibians than previously thought and thus contribute to the prevalence of homomorphic sex chromosomes in this group.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.